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PKA-Chromatin Association at Stress Responsive Target Genes from Saccharomyces Cerevisiae

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PKA-Chromatin Association at Stress Responsive Target Genes from Saccharomyces Cerevisiae View metadata, citation and similar papers at core.ac.uk brought to you by CORE Published in %LRFKLPLFDHW%LRSK\VLFD$FWD %%$ *HQH5HJXODWRU\0HFKDQLVPV ± provided by RERO DOC Digital Library which should be cited to refer to this work. PKA-chromatin association at stress responsive target genes from Saccharomyces cerevisiae Leticia Baccarini a, Fernando Martínez-Montañés c, Silvia Rossi a, Markus Proft b, Paula Portela a,⁎ a Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, IQUIBICEN-CONICET, Universidad de Buenos Aires, Buenos Aires, Argentina b Instituto de Biomedicina CSIC, Valencia, Spain c Department of Biology, University of Fribourg, Fribourg, Switzerland Gene expression regulation by intracellular stimulus-activated protein kinases is essential for cell adaptation to environmental changes. There are three PKA catalytic subunits in Saccharomyces cerevisiae: Tpk1, Tpk2, and Tpk3 and one regulatory subunit: Bcy1. Previously, it has been demonstrated that Tpk1 and Tpk2 are associated with coding regions and promoters of target genes in a carbon source and oxidative stress dependent manner. Here we studied five genes, ALD6, SED1, HSP42, RPS29B,andRPL1B whose expression is regulated by saline stress. We found that PKA catalytic and regulatory subunits are associated with both coding regions and promoters of the analyzed genes in a stress dependent manner. Tpk1 and Tpk2 recruitment was completely abolished in cat- alytic inactive mutants. BCY1 deletion changed the binding kinetic to chromatin of each Tpk isoform and this strain displayed a deregulated gene expression in response to osmotic stress. In addition, yeast mutants with high PKA activity exhibit sustained association to target genes of chromatin-remodeling complexes such as Snf2-catalytic subunit of the SWI/SNF complex and Arp8-component of INO80 complex, leading to upregulation of gene expression during osmotic stress. Tpk1 accumulation in the nucleus was stimulated upon osmotic stress, while the nuclear localization of Tpk2 and Bcy1 showed no change. We found that each PKA subunit is transported into the nucleus by a different β-karyopherin pathway. Moreover, β-karyopherin mutant strains abolished the chromatin association of Tpk1 or Tpk2, suggesting that nuclear localization of PKA catalytic sub- units is required for its association to target genes and properly gene expression. 1. Introduction slow respiratory growth or stationary phase [9]. There are three PKA catalytic subunits in S. cerevisiae: Tpk1, Tpk2, and Tpk3 and one regulatory Signal transduction pathways play an important function in proper subunit: Bcy1 [10,11]. cellular response against extracellular injuries. Cellular adaptation to PKA signaling pathway controls the expression of several genes, http://doc.rero.ch environmental changes involves a strict regulation of gene expression. both positively and negatively. PKA is known to inhibit Rim15, a positive In eukaryotic cells, stress-activated protein kinases (SAPKs) have an regulator of the transcription factor Gis1, which induces genes required essential role in adaptation to an extracellular stimulus [1]. for postdiauxic growth [12], PKA also inhibits the transcription factors In response to stress or nutritional conditions, protein kinases can Msn2/4, which induces general stress responsive gene expression [13]. regulate gene expression by association to chromatin both in promoters Nuclear localization of Msn2/4 is controlled by PKA [14]. On the other and/or actively transcribed regions and further phosphorylation of hand, it has been described that PKA negatively regulates the activity transcription factors, histones, chromatin-modifying complexes, and/ of the RNA polymerase II machinery by direct phosphorylation of or the transcription machinery. In Saccharomyces cerevisiae protein Srb9, a component of the Srb Mediator complex [15], and also promot- kinases such as Hog1 [2,3], Fus3, Kss1, Tor1 [4], Sch9 [5],andPKA[6,7] ing the accumulation of inactive RNA Pol II at ribosome biogenesis genes as well as Schizosaccharomyces pombe Sty1 [8] have been described as [16]. Recently, it has been described that in response to glucose, PKA chromatin-associated kinases. phosphorylates the transcription factor Rgt1 promoting its dissociation Yeast PKA acts positively on phenotypes associated with rapid from HXT promoters, resulting in the upregulation of HXT genes [17].Fi- fermentative growth and negatively on phenotypes associated with nally, PKA controls FLO11 gene expression not only positively by Tpk2 [18,19], but also negatively via Tpk1 through the kinase Yak1 [20]. Genome wide ChIP-on-Chip experiments in S. cerevisiae revealed ⁎ Corresponding author at: Laboratorio de Biología Molecular y Transducción de that Tpk1 and Tpk2 interacted with chromatin. Tpk1 was found physi- Señales, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales — UBA, Ciudad Universitaria Pabellón II Piso 4, Buenos Aires, Argentina. cally associated with genes that are actively transcribed during glucose E-mail address: [email protected] (P. Portela). growth or under low oxidative stress. Tpk2 was found mainly associated 1 with the promoter regions of ribosomal protein genes under oxidative CCCAAC3′ and Rev: 5′GATTCCGACCTTGTTTGGAGC3′. TPK2 gene: For 5′ stress. Occupancy of the Tpk3 subunit was not detected due its low pro- ACTGCAGATTTGACAAGAAG3′ and Rev: 5′GTGCGCCAGATTTTGGTGGA tein expression level [6]. 3′. BCY1 gene: For 5′AGACCATGATTATTTCGGTG3′ and Rev: 5′GTAGTA Subcellular localization of PKA subunits is strongly regulated by nu- ACAGCAGTAGTAGA3′SNF2 gene: For 5′ACTTCAAGCGTGGCTGAATC3′ trient availability and stress conditions. In exponentially glucose- and Rev: 5′CATCCCAACTCGGTTAATGG3′. ARP8 gene: For 5′CGTGATAT growing cells, Bcy1 and Tpk2 localization is mainly nuclear, whereas GAATCCCGCTCT3′ and Rev: 5′TGCTTCGTTGATGTCTGCAA3′ employing Tpk1 and Tpk3 show a nucleus-cytoplasmic distribution [21,22]. Cell genomic DNA from the corresponding strain (see Table 1). DNA in stationary phase of growth or stressed cells show re-localization of sequence was confirmed by sequencing and expression of tagged protein all PKA subunits towards the cytoplasm, where Tpk2 and Tpk3, in par- was monitored by western-blot and fluorescence accordingly. Strains ticular, are accumulated in RNP (RiboNucleoProtein) granules [21,23]. were grown in rich medium containing 2% bactopeptone, 1% yeast The traffic of RNA and protein between nucleus and cytoplasm is extract, and 2% glucose (YPGlu) at 30 °C. Solid media contained 2% agar. mediated by specialized carriers that translocate these macromolecules across the nuclear membrane. The budding yeast S. cerevisiae has 14 2.1.1. Osmotic stress, expression levels, and cell viability members of the β-karyopherin family which are classified into Osmotic stress was performed by addition of 0.4 M NaCl. Aliquots importins and exportins, depending on whether they transport the were taken at different times and processed according to each determi- cargo in or out of the nucleus. Most β-karyopherins directly bind the nation. The zero time point corresponds to samples taken immediately NLS (Nuclear Localization Signals) of their cargos. The importin-β path- before NaCl addition. Expression level analysis: crude extracts of the way (Kap95 in yeast) recognizes the classical NLS through the adaptor strains indicated on each graph were prepared according to Materials protein importin-α (Kap60 in yeast). The β-karyopherins Kap108, and methods, and were subjected to SDS/PAGE, Western blotting Kap120, Kap123, and Kap114 are functionally redundant and act as using an anti-GFP antibody (Supplementary Fig. S1A) or anti-TAP anti- importins or exportins [24–26]. Not only the β-karyopherins can recog- body (Supplementary Fig. S3A) and quantified by densitometry as de- nize more than one cargo and potentially also more than one NLS, but scribed below. The expression levels of each tagged protein in also one cargo can be recognized by more than one β-karyopherin different strains for each condition were expressed relative to the ex- [27,28]. The nuclear-cytoplasmic transport of proteins is a flexible pression levels under normal condition. The bars represent the mechanism that responds to nutrient availability and stress [29,30]. mean ± SEM from two independent experiments. Cell viability: strains The β-karyopherin pathway responsible of the nuclear trafficof were grown to exponential phase at 30 °C and treated with 0.4 M NaCl S. cerevisiae PKA subunits has not been previously analyzed. during 40 min. Cell viability of wild type and mutant strains after NaCl Here we determined that Tpk1 accumulates in the nucleus post- treatment was verified by spot assay method (Supplementary Fig. S1B osmotic stress, however Tpk2 and Bcy1 did not change their nucleus- and Supplementary Fig. S3B). cytoplasmic distribution, being preferentially nuclear. We found that each PKA subunit is actively transported into the nucleus by different 2.1.2. Osmosensitivity assay β-karyopherins pathways. We found that PKA subunits associate with Serial dilutions of exponentially growing cells on YPGlu were spot- different gene regions in response to osmotic stress. Catalytically inac- ted directly onto the plates containing different NaCl concentrations. tive versions of Tpk1 and Tpk2 do not associate with the analyzed Plates were photographed after 2 days at 30 °C. gene regions. A strain with a deletion of the BCY1 gene showed a higher Tpk1 recruitment in comparison
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